This invention relates to plasminogen activators which are useful thrombolytic agents. More particularly, this invention relates to a modified tissue plasminogen activator having an improved in vivo half-life.
It is known that various plasminogen activators (PA) are widely distributed throughout the body and can be purified from tissue extracts. Typical examples of tissue sources are kidney, lung and uterus tissues. The best characterized of these plasminogen activators fall into two major groups, urokinase plasminogen activator (u-PA) and tissue plasminogen activator (t-PA). u-PA and t-PA are present in ng/ml concentrations in human plasma but are immunologically unrelated. t-PA has been demonstrated to have higher affinity for fibrin than u-PA. u-PA products isolated and purified from human urine and from mammalian kidney cells are pharmaceutically available as thrombolytic agents.
Due to the extremely low concentration of t-PA in blood and tissue extracts, other sources and means of producing this preferred thrombolytic agent have been sought after.
One method of producing t-PA on a large scale comprises isolating the protein from the culture fluid of human melanoma cells grown under in vitro cell culture conditions. An established human melanoma cell line (Bowes) has been used for this purpose. See, for example, European Patent Application 41,766, published Dec. 16, 1981; Rijken and Collen, J. Biol. Chem. 256(13), 7035-7041 (1981); and Kluft et al., Adv. Biotech. Proc. 2, Alan R. Liss, Inc., 1983, pp. 97-110. The Bowes melanoma t-PA is a glycoprotein which has a molecular weight of about 68,000-70,000 daltons and a 527 amino acid structure with serine at the NH.sub.2 -terminus. The melanoma t-PA can exist as two chains, an A-chain and a B-chain. It also separates into two variants (or isoforms) in the A-chain, known as types I and II, which differ by about M.sub.r 2000-3000. See Ranby et al., FEBS Lett. 146 (2), 289-292 (1982), and Wallen et al., Eur. J. Biochem. 132, 681-686 (1983). Type I is glycosylated at Asn-117, Asn-184 and Asn-448 whereas Type II is glycosylated only at Asn-117 and Asn-448 according to Pohl et al., Biochemistry 23, 3701-3707 (1984). A high mannose structure has been assigned to Asn-117 whereas two complex carbohydrate structures are assigned to Asn-184 and Asn-448 by Pohl et al., "EMBO Workshop on Plasminogen Activators," Amalfi, Italy, Oct. 14-18, 1985. Genetic information from the Bowes melanoma cell line also has been embodied in E. coli by conventional recombinant DNA gene splicing methods to permit the production of the t-PA protein moiety by that microorganism. See, for example, UK Patent Application 2,119,804, published Nov. 23, 1983; Pennica et al., Nature 301, 214-221 (1983); and Vehar et al., Bio/Technology 2 (12), 1051-1057 (1984). Recombinant t-PA produced by the expression of Bowes melanoma genetic material in cultured mammalian cells has been administered to humans with some measure of effectiveness. See Collen et al., Circulation 70(16), 1012-1017 (1984).
The recombinant-derived t-PA produced in E. coli is non-glycosylated and contains only the protein moiety of t-PA. Although the specific function of the carbohydrate moiety on t-PA has not been determined, it is known, in general, that glycosylation can cause certain differences of which the following are of biological interest: antigenicity, stability, solubility and tertiary structure. The carbohydrate side-chains also can affect the protein's half-life and target it to receptors on the appropriate cells. See, for example, Delente, Trends in Biotech. 3 (9), 218 (1985), and Van Brunt, Bio/Technology 4, 835-839 (1986). The functional properties of carbohydrate-depleted t-PA are further discussed by Little, et al., Biochemistry 23, 6191-6195 (1984), and by Opdenakker et al., "EMBO workshop on Plasminogen Activators," Amalfi, Italy, Oct. 14-18, 1985. The latter scientists report that enzymatic cleavage of carbohydrate side-chains from the melanoma (Bowes) derived t-PA by treatment with .alpha.-mannosidase causes an increase in the biologic activity of the modified t-PA.
Cultured normal human cells also have been used as a source of t-PA as can be seen from U.S. Pat. Nos. 4,335,215, 4,505,893, 4,537,860, and 4,550,080. Various cell sources mentioned in said patents are primary embryonic (or fetal) kidney, lung, foreskin, skin and small intestines (Flow Laboratories) or the AG1523 cell line. Brouty-Boye et al., Bio/Technology 2 (12), 1058-1062 (1984), further disclose the use of normal human embryonic lung cells for the production of t-PA. Rijken and Collen, J. Biol. Chem. 256(13), 7035-7041 (1981), and Pohl et al., FEBS Lett. 168(1), 29-32 (1984), disclose the use of human uterine tissue as a t-PA source material. European Patent Application 236,289, published Sep. 9, 1987, describes a uniquely glycosylated t-PA derived from normal human colon fibroblast cells.
Production of glycosylated t-PA in non-human mammalian cells also is known. Thus, Kaufman et al., Mol. Cell. Biol. 5, 1750-1759 (1985), and European Patent Application 117,059, published Aug. 29, 1984, describe the use of Chinese hamster ovary cells and Browne et al., Gene 33, 279-284 (1985), describe the use of mouse L cells for such production. Kaufman et al., state that the Chinese hamster ovary t-PA is glycosylated in a similar but not identical manner as native t-PA. Glycosylated forms of t-PA obtained by recombinant DNA are further described by Zamarron et al., J. Biol. Chem. 259 (4), 2080-2083 (1984), and Collen et al., J. Pharmacol. Expertl. Therap. 231 (1), 146-152 (1984).
Production of glycosylated t-PA by recombinant DNA yeast cells also has been reported. Thus, European Patent Application 143,081, published May 29, 1985, describes a recombinant yeast plasmid vector which encodes human t-PA from Hela cells. European Patent Application 174,835, published Mar. 19, 1986, describes a t-PA with selected glycosylation expressed in yeast in which the cDNA encoding for the t-PA is derived from Bowes melanoma. European Patent Application 178,105, published Apr. 16, 1986, discloses a glycosylated uterine t-PA expressed in yeast cells or mouse C-127 cells. In the latter case, a bovine papilloma virus is used as the vector.
Notwithstanding the great variety of sources for obtaining t-PA, one of the problems that exists with the normal t-PA molecule is its relatively short half-life. Intravenously administered t-PA disappears rapidly from the circulation into the liver where it is degraded. The half-life of this clearance is approximately 2 minutes in rabbits [Korninger et al., Thromb. Haemostas. 46, 658-661 (1981)]. Recent clinical studies have suggested that the half-life in humans may be slightly longer, on the order of 3-4 minutes [Nilson et al., Scand. J. Haematol. 33, 49-53 (1984)]. Since thrombolysis in vivo takes, at best, several hours to achieve, these findings indicate that the successful application of t-PA for thrombolysis in man will require its continuous infusion. Development of a t-PA with a longer half-life would allow for shorter periods of administration or a smaller dose.
Recently, so-called second generation type t-PAs have been prepared by recombinant DNA technology and various protein engineering schemes in attempting to improve the t-PA molecule. It is known that the normal t-PA molecule has five functional domains or regions: A fibronectin-like finger domain F); an epidermal growth factor region (GF); two kringle regions (K1 and K2); and a serine protease region (SP). In the 527 amino acid sequence of the normal t-PA molecule described by Pennica et al., Nature 301, 214-221 (1983), the finger region comprises residues 1-43; the growth factor region comprises residues 44-91; kringle refers to a characteristic triple disulfide structure of which t-PA has two such regions, K1--residues 92-173, and K2--residues 180-261; and the serine protease comprises residues 262-527. The SP catalytic site is formed from the His-322, Asp-371 and Ser-478 residues. Various deletions of one or more of these regions together with elimination of one or more of the glycosylation sites such as by site-directed mutagenesis have been described heretofore. See, for example, Kagitani et al., FEBS Lett 189(1), 145-149 (1985); Zonneveld et al., Proc. Natl. Acad. Sci. USA 83, 4670-4674 (1986); Verheijen et al., The EMBO J. 5 (13), 3525-3530 (1986); Ehrlich et al., Fibrinolysis 1, 75-81 (1987); Klausner, Bio/Technology 4, 706-710 (1986) and 5, 869-870 (1987); and various abstracts in Thromb. Haemostasis. 58, 1-676 (1987). European Patent Applications 234,051, published Sep. 2, 1987, and 242,836, published Oct. 28, 1987, and PCT International Application WO 87/03906, published Jul. 2, 1987, disclose a variety of t-PA mutants having alterations in the arrangement of one or more of the functional domains.
Specific examples of t-PA having such domain changes are as follows:
In European Patent Application 196,920, published Oct. 8, 1986, a modified t-PA is described which has the intact B chain of mature t-PA linked to kringle K2 as the only functionally and structurally intact domain of native t-PA A chain. European Patent Application 207,589, published Apr. 2, 1986, describes a t-PA in which all or part of the growth factor region (GF) has been deleted. Japanese Patent KOKAI No. 48378/87, laid open Mar. 3, 1987, discloses an improved t-PA having a part or the whole of the kringle domains deleted. In European Patent Application 213,794, published Mar. 11, 1987, a hybrid t-PA is described which has a plurality of heterologous kringles (2 to 6 kringles), for example, a t-PA protein with a prothrombin or urokinase kringle region. In PCT Inter. Appln. WO 87/03906, several modified t-PAs are disclosed, including one in which the growth factor region (GF) and the first kringle region (K1) have been deleted but which contains an additional second kringle region (K2).
Specific examples of t-PA having various site-directed mutagenesis are as follows:
In European Patent Application 178,105, published Apr. 16, 1986, a modified t-PA is described in which one or more of the glycosylation sites have been eliminated by site-directed mutagenesis of Asn to Gln at the glycosylation sites in the kringle and serine protease regions. The amino acid residues Asn-120, -187 and -451 in the described uterine t-PA are equivalent to residues Asn-117, -184 and -448, respectively, in the Bowes melanoma t-PA. U.K. Patent Application G.B. 2,173,804, published Oct. 22, 1986, describes mutagenesis in the region of residues 270 to 279 to prevent conversion to the two-chain form of t-PA, especially mutagenesis of Arg-275 and Ile-276, e.g. Arg-275 .fwdarw. Gly or Glu. In PCT International Application WO 86/01538, published Mar. 14, 1986, the mutant Lys-227 .fwdarw. Ile is described. A variety of site-mutagens are also described in European Patent Application 227,462, published Jul. 1, 1987, including mutagenesis at the above glycosylation sites and at the cleavage sites in the region 272-280, especially in the sequence Phe(274)-Arg(275)-Ile(276)-Lys(277). Other such mutants are disclosed in European Patent Application 238,304, published Sep. 23, 1987.